INVESTIGADORES
GONZALEZ LEBRERO Rodolfo Martin
congresos y reuniones científicas
Título:
Pre-steady kinetic of ATP hydrolysis by dengue virus NS3 helicase in single turnover conditions
Autor/es:
RODOLFO M GONZÁLEZ LEBRERO; LG GEBHARD; AV GAMARNIK; SB. KAUFMAN
Lugar:
Salta
Reunión:
Congreso; XXXIX Annual Meeting of the Argentinean Biophysical Society (SAB) and 3rd Latin American Protein Society Meeting; 2010
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
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The NS3 dengue virus helicase is a
molecular motor protein that unwinds duplex RNA during the
replication of the viral genome. The binding and hydrolysis of
adenosine triphosphate (ATP) are essential for helicase function.
However, the reaction mechanism that explains the coupling between
nucleotide hydrolysis and duplex RNA unwinding is still unknown.
Thus, we propose to study the kinetic mechanism of nucleotide
hydrolysis during the catalytic cycle of NS3. According to this, we
performed pre-steady state experiments of ATP hydrolysis by following
orthophosphate(Pi) release in single turnover conditions. The time
courses of Pi release were obtained at different concentrations of
ATP and NS3 but, in all cases, with excess of enzyme on substrate and
pseudo-first order conditions. When the enzyme concentration was
1.022 µM and [ATP] = 0.2, 0.051 and 0.00019 µM the time courses of
Pi release were monoexponential curves with apparent rate contants:
0.048, 0.048 and 0.053 s-1 respectively. When the enzyme
concentration was decreased to 0.050 and 0.014 µM with [ATP] =
0.00019 and 0.00016 µM also monoexponential curves were obtained
with apparent rates constants: 0.0054 and 0.0019 s-1 respectively.
These results suggests that, in the present experimental conditions
the rate limiting step of ATP hydrolysis is the formation of
collisional complex NS3-ATP with an apparent second order rate
constant of 0.0466 µM-1 s 1. NS3 protein was cloned from the
dengue virus 2 16681 infectious clone. The recombinant protein was
purified by histidine tag affinity chromatography. All time
courses were carried-out at 25 °C in media containing: 100 mM KCl;
25 mM MOPS/K (pH=6.5); 2 mM MgCl2; 0.5 mM EDTA; 3% glycerol.